The present invention relates to particle detection devices, particularly to devices for single-particle detection and to methods for using the devices to achieve extremely high sensitivity detection of specific particles, and more particularly to devices for affinity-based electrochemical detection of particles with single-particle sensitivity, and wherein a single device or an array of devices employing surface-attached specific affinity components may be used to selectively capture particles.
Various approaches to detection of particles in a solution have been developed over the years. Molecular Recognition Materials (MRMs) capable of selective binding to a particle are used in a wide range of assays and sensors. Such materials include Antibodies (Abs) and natural receptor ligands for various biological particles, but may also include a range of synthetic MRMs including combinatorial peptides and other combinatorially produced materials and designed ligands and chelators. Abs may be considered the archetypal of MRMs, and are used in an enormous range of assays and sensors. Assays using MRMs may be divided according to whether they are homogenous (all reagents are mixed into a single phase and the result is read) or heterogeneous. In this second category, MRMs are generally immobilized onto a surface and then exposed to a solution containing particles, as described above. Commonly used formats include the ELISA assay and formats such as latex bead agglutination assays, but an enormous variety of implementations exists. These methods may be used for the range of particle types defined here (bacteria through molecules), but generally require a large number of the particles to be present. This is because affinity-based methods such as ELISA assays generally work on a continuum basis, rather than a statistical (i.e., single particle) basis.
For the detection and identification of bacteria or bacterial spores, viruses, or other pathogens, classical microbiological methods (in vitro and in vivo culture on selective media, colony morphology, chemical staining) still predominate. In the past two decades, the use of MRMs in the form of immunohistochemistry, coupled with light (i.e., fluorescence) or electron microscopy, has created alternative means of pathogen identification. Such methods may be adapted for automated identification of organisms through means such as fluorescence-based flow cytometry. More recently, Polymerase Chain-Reaction (PCR) methods have been developed that allow determination of whether a particular organism is present or not. PCR requires isolation of nucleic acids from the sample, and many cycles of reaction, typically taking on the order of 30 minutes or longer.
Both culture and PCR have the advantage that, in principle, even a single organism in a sample may be detached, although the amplification of material (through growth of organisms, or repeated reactions) that allows this required time. Formats such as flow cytometry, where each particle might be labeled with a selectively-binding fluorescent Abs, do permit rapid single particle detection, albeit generally in a rather large package. The problem is that the method has no way of confirming if a given single particle was actually the target analyte, or something to which the Abs bound through non-specific binding (NSB) interactions. To rule out NSB requires a secondary test, by means such as PCR or dissociating the Abs from the particle, re-labeling with a second MRM, and sending it through the flow cytometer system again while such systems are possible to implement, they compound the complexity, size, time, and reagent requirements of the original methods. There are also limitations in throughput for all of these methods, in that detecting a single particle in a given volume requires somehow processing at least that much volume (preferably several times the volume to allow for a given statistical level of surety). Again, although it is in principle possible to address these difficulties through scaling (multiple independent channels operating simultaneously), this can only be done at the cost of greater instrumental complexity.
Another concept that needs to be introduced is that it is advantageous if the process of measuring the presence of the particle does not destroy it. This then facilitates repeated measurements of the particle's properties, which can be used to confirm its presence and identity. For example, in flow cytometry, once the fluorescently-labeled particle passes through the laser-based fluorescence detection portion of the cytometer, the fluors are generally destroyed (through photo-bleaching), so that particle cannot be re-assayed without stripping off the (rather tightly bound) Ab labels and re-labeling. Fluorescence measurement of a natively fluorescent particle (i.e., molecules) may result directly in photodestruction of the particle. Direct electrochemical detection also generally results in chemical alteration of the analyte.
For the particular case of detection of bacterial spores (of great interest for anti-biological warfare/counter-terrorism applications), classical microbiological methods, ELISAs, Ab-flow cytometry, and PCT have been applied, with their attendant advantages and limitations. Various methods have been developed that either do not require MRMs, or use other transduction mechanisms. Rosen, et al., Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photo-Luminescence, Analytical Chemistry 89 (1997) 1082–1805, have reported that when endospores are incubated with terbium chloride, a photoluminescent complex is formed with spore case calcium dipicolinate. After filtration, a detection limit of 4.4×105 C.F.U./mL is obtained. Gatto-Menking, et al., Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor, Biosensors and Bioelectronics 10 (1995) 501–507, have developed an immunomagnetic sensor for biotoxoids that uses a commercial electrochemiluminescence analyzer (ORIGEN®). A spore detection limit of 100 has been reported using this method.
Electrode arrays have been a topic of interest as miniature sensors for twenty years. In summary, these devices use microfabricated electrode arrays to detect a variety of environmental and biological compounds. These patents all use direct coulometric detection of the analyte by constructing on-chip electrical circuits to monitor individual currents directly as a function of time. A range of other electronic sensors, coupled with MRMs, have been proposed for pathogen detection wherein a change in the impedance signature of the sensors element occurs when particles bind to the MRM. These include, in particular, capacitance and impedance-based measurements on microelectrodes, and miniature oscillators of various kinds. Although the sensor element we describe does depend for its operation on the general concept of a change in the impedance of the element when the particle binds to the MRMs, it is fundamentally different, in that it operates on a single particle, statistical basis, and has a microscopic structure that makes this possible.
From the above summary it appears that the desirable properties for an improved sensor design are: 1) the ability to work at the statistical limit of sensitivity, 2) single-step operation, 3) continuous operation, 4) simplified reagents and fluidic systems, 5) ease of scaling to improve throughput, 6) low consumption of power and reagents, 7) non-destructive measurement of the particle.
The present invention provides considerable advantages of the prior known approaches to particle detection. The sensor element or device of the present invention has been demonstrated to respond to a single particle. The devices and methods of the invention do not require a prior or subsequent labeling step of the particle, though there are methods for use of the devices where such steps might be incorporated with some advantage. The detector devices of this invention represents a substantial improvement over electrode array detectors using traditional potentiostatic control/monitoring by simplifying the current measurement apparatus and allowing an increase in the information density of the detector. Their mode of operation allows single particle tracking and real-time monitoring of thousands to millions of active elements on a miniature device. It is not feasible to construct a million element electrode array and monitor current with a million potentiostats in a small device. The particle detector and method of the present invention employ a surface-attached specific affinity components to selectively capture a particle on elements of an array of single particle sensors. The particle detector array may contain a very large number of elements, or only single detector elements may be used. In addition, the invention includes a capacitive read-out circuit for electrochemical measurements which is more effective to the conventional potentiostatic control circuit previously used in electrochemical measurements.